Imaging method and device for carrying out said method

ABSTRACT

The invention relates to an imaging method for simultaneously determining in vivo distributions of bioluminescent and/or fluorescent markers and radioactive markers at identical projection angles, the distribution of the bioluminescent and/or fluorescent markers being determined by separate detection of photons having a first average energy, which are emitted by the bioluminescent and/or fluorescent markers, by means of at least one first detector and the distribution of the radioactive markers being determined by simultaneous separate detection of photons having a second average energy, which are emitted by the radioactive markers, by means of at least one second detector. Furthermore, it also relates to an apparatus for carrying out the imaging method, containing at least one CCD camera ( 1, 2 ) as first detector, at least one single photon emission computer tomography (SPECT) detector ( 3 ) as second detector and a layer ( 5 ), which essentially reflects the photons of the bioluminescent and/or fluorescent markers and essentially transmits the photons of the radioactive markers.

The invention relates to an imaging method for determining in vivodistributions of bioluminescent, fluorescent and radioactive markers.Furthermore, it relates to an apparatus for carrying out the imagingmethod.

The qualitative and quantitative detection of morphological, functionaland biochemical parameters using imaging methods is the basis of diversefields of medical research and application. Known imaging methods usedin tumor research, inter alia, are, by way of example, single photonemission computer tomography (SPECT) using radionuclides or opticaltechnologies using fluorescence or bioluminescence.

In the case of single photon emission computer tomography, radioactivemarkers are injected into the animal or human to be examined, whichmarkers concentrate in specific organs or tissue types of theanimal/human depending on the biological carriers which transport them.The radioactive markers emit radioactive radiation (γ radiation), theintensity of which in a specific region of the subject to be examineddepends on the concentration of the marker in said region. Theradioactive radiation is detected by means of a γ camera orscintillation camera. For carrying out high-resolution studies on smalllaboratory animals, apparatuses for detecting the γ photons aredisclosed for example in D. P. McElroy et al.: Evaluation of A-SPECT: ADesktop Pinhole SPECT System for Small Animal Imaging, Proc. Med. Imag.Conf. San Diego 2001, M10-4 or in A. G. Weisenberger et al.: Developmentof a Novel Radiation Imaging Detector System for In Vivo Gene Imaging inSmall Animal Studies, IEEE Transactions on Nuclear Science, Vol. 45, No.3 (June 1998) 1743-1749. Examples of fields of application for suchtomographs are preclinical research or radiotracer development.

A further imaging method known in the prior art for in vivo examinationis an optical method that makes use of fluorescent or bioluminescentmarkers. The latter serve for example as reporter genes which are usedfor observing gene expression since the associated proteins produce ameasurable optical signal. The gene that codes for the proteinluciferase is a much used reporter gene. In this case, the gene for aspecific protein is replaced by the luciferase gene. Upon activation ofthe associated promoter, the latter then drives the transcription of theluciferase gene. The enzyme luciferase from the North American fireflyPhotinus pyralis catalyzes the oxidative decarboxylation of luciferin inthe presence of ATP and Mg²⁺. This gives rise to flashes of light whichaccumulate to form the light emission from these animals. Theluminescence occurring in the presence of luciferin and ATP consequentlyindicates the expression of luciferase. This optical signal can easilybe measured, for example by means of CCD cameras. In the same way asbioluminescent reporter genes, fluorescent reporter genes are used aswell, for example the gene for the green fluorescent protein (GFP). Suchproteins are stimulated into fluorescence by irradiation with anexternal light source. In vivo imaging of gene expression by opticalmethods is disclosed for example in C. Bremer, R. Weissleder: In VivoImaging of Gene Expression: MR and Optical Technologies, AcademicRadiology, Vol. 8, No. 1 (January 2001) 15-23. These methods are alsoused, inter alia, for in vivo tumor monitoring (R. Weissleder et al.: InVivo Imaging of Tumors with Protease-Activated Near-Infrared FluorescentProbes, Nature Biotechnology, Vol. 17 (April 1999) 375-378).

Advantages of the molecules which emit the optical (fluorescence orluminescence) photons are, inter alia, that they have specific chemicalproperties of medical interest that are lacking in the radioactivelymarked components, for example that they can be activated by specificenzymatic interactions. By contrast, the radioactive substances have theadvantage that the γ rays that they emit have much lower probabilitiesof interaction with dense tissue than the optical photons, so that theycan penetrate through large tissue volumes or thicknesses. Furthermore,their slight or lack of interaction with the biochemical properties ofthe tissue to be examined may be advantageous. In the prior art, bothimaging methods, the method for detecting the optical photons emitted bythe fluorescent or luminescent molecules, on the one hand, and themethod for detecting the higher-energy photons emitted by theradioisotopes, on the other hand, are carried out separately, indifferent apparatuses. A comparison of the images obtained by the twoimaging methods is possible only to a limited extent since they cannotbe obtained simultaneously and at the same projection angles. Theproblems of excessive burdening of the subject to be examined, thenon-reproducibility of kinetic studies, the non-identical imaginggeometries, animal and organ movement and the correct superposition ofthe images arise when the two methods are carried out successively.

Therefore, the present invention is based on the object of avoiding thedisadvantages of the prior art and of combining the advantages of thetwo technologies described above.

This object is achieved by means of an imaging method for simultaneouslydetermining in vivo distributions of bioluminescent and/or fluorescentmarkers and radioactive markers at identical projection angles, thedistribution of the bioluminescent and/or fluorescent markers beingdetermined by separate detection of photons having a first averageenergy, which are emitted by the bioluminescent and/or fluorescentmarkers, by means of at least one first detector and the distribution ofthe radioactive markers being determined by simultaneous separatedetection of photons having a second average energy, which are emittedby the radioactive markers, by means of at least one second detector.

In this connection, projection is to be understood as thetwo-dimensional imaging of a three-dimensional energy distribution at aspecific projection solid angle of the detector with respect to theobject to be imaged. In the invention, the projection solid angles ofthe two detector systems with respect to the object are identical, thatis to say that the object is “viewed” by both detectors from anidentical projection angle.

Optical photons of the bioluminescent and/or fluorescent markers have a(first) average energy in the range between 1 eV and 3 eV. Photons ofthe radioactive markers have a second average energy in a range between10 keV and 600 keV. The imaging method according to the invention iscarried out in vivo. It may be applied to living laboratory animals, byway of example. In this case, it is advantageously possible

-   -   to observe transport, metabolism and excretion of active        substances in the living organism, and    -   to measure biological processes in their natural environment.

In a preferred embodiment of the present invention, the photons of thebioluminescent and/or fluorescent markers having the first averageenergy and the photons of the radioactive markers having the secondaverage energy are separated for the separate detection with the aid ofa layer, the layer essentially reflecting or transmitting the photons ina manner dependent on their energy. By way of example, γ radiation istransmitted without or with only slight interactions with the layer,while the lower-energy optical radiation is reflected by the layer. Thisenables separate detection of the photons having different energieswhich are emitted at the same projection angle by markers in the subjectto be examined.

In a preferred embodiment of the present invention, the layer serves forreflecting the photons of the bioluminescent and/or fluorescent markersin the direction of the at least one first detector and for transmittingthe photons of the radioactive markers in the direction of the at leastone second detector. Consequently, the photons having different energiesare not only separated by the layer but are also already “steered” inthe direction of the detectors that detect them.

Preferably, the bioluminescent and/or fluorescent markers comprise atleast one marker from the group consisting of the markers of theluciferase reporter, the marker molecules having emission wavelengths inthe near infrared range (NIRF molecules) and the molecules of the GFP(green fluorescent protein). Markers from this group have already beensuccessfully integrated in the reporter gene concept and detected invivo (in living) animals. The luciferase produces a blue or yellow-greenlight in the context of an enzymatic reaction (see above). The enzymesubstrates that form the starting substances for the light-emittingproducts are called luciferins. Luciferase/luciferin systems are foundfor example in the firefly Photinus pyralis (emission maximum at 562nm), in fireflies and in numerous luminous marine bacteria (emissionmaximum at 489 nm).

The green fluorescent proteins (GFP) which are characterized the bestoriginate from the Pacific jellyfish Aequorea victoria and the sea pansyRenilla reniformis. In both cases, the GFP transforms bluechemiluminescence into green fluorescence (emission maximum at 508 μm).GFP is a relatively small protein, consisting of 238 amino acids.

The molecules having emission wavelengths in the near infrared range(NIRF markers) have lower probabilities of interaction in the tissuethan photons having wavelengths in the visible wavelength spectrum andtherefore a greater penetration depth.

By way of example, proteins, lipids, RNA or DNA can be marked by thebioluminescent and/or fluorescent markers or by the radioactive markers.

Preferably, the radioactive markers comprise at least one marker fromthe group As-72, Br-75, Co-55, Cu-61, Cu-67, Ga-67, Gd-153, I-123,I-125, I-131, In-111, Ru-97, Tl-201, Tc-99m and Xe-133. The radioisotoperespectively used is selected as a marker with regard to its half-lifeand the energy of the radiation that it emits, in a manner dependent onthe biological process to be measured.

In a preferred embodiment of the present invention, the detection of thephotons having the first average energy is carried out by means of atleast one CCD camera and the detection of the photons having the secondaverage energy is carried out by means of at least one single photonemission computer tomography (SPECT) detector comprising a collimatorwith at least one aperture.

CCDs (charge coupled devices) are charge coupled imaging sensors thatserve for highly sensitive detection of photons. The CCD camera isdivided into a multiplicity of small light-sensitive zones (pixels)which produce the individual points of an image. The grid of pixels isformed by a circuit structure on a semiconductor crystal (usuallysilicon). The method of operation of the CCD camera is based on theliberation of electrons by impinging light in the semiconductormaterial. A photon falling onto a pixel liberates at least one electronthat is held fixed by an electrical potential at the location of thepixel. The number of electrons liberated at the location of the pixel isproportional to the intensity of the light incident at said location.The number of electrons is measured in each pixel, with the result thatan image can be reconstructed. CCDs should be cooled since otherwisemore electrons would be read out which would not be liberated as aresult of the light incidence but rather as a result of heating. In thecase of the present invention, the optical photons of the bioluminescentand/or fluorescent markers are preferably detected with the aid of atleast one CCD camera.

SPECT detectors usually contain γ or scintillation cameras. Ascintillator absorbs the γ rays emitted by the radioisotopes. As aresponse thereto, the scintillator emits light scintillations comprisingvisible light which are detected by a group of photo-multipliers of theSPECT detector and converted into a measurable electrical signal. Thelocation of the radioactive emission of photons in a body can belocalized only when a collimator is arranged between the body and thescintillator. Said collimator serves for shielding from the scintillatorphotons which are not situated in an acceptance region defined by thecollimator geometry. Furthermore, the collimator defines the field ofview of the detector system.

The present invention furthermore relates to an apparatus for carryingout the imaging method according to the invention. Said apparatuscontains at least one cooled CCD camera as first detector, at least onesingle photon emission computer tomography (SPECT) detector as seconddetector and a layer, which essentially reflects the photons of thebioluminescent and/or fluorescent markers and essentially transmits thephotons of the radioactive markers. The layer serves (as alreadymentioned above) for separating the photons having different energieswhich are detected in different detectors (CCD camera and SPECTdetector). The SPECT detector preferably comprises a collimator, ascintillator and a multiplicity of photomultipliers with associatedelectronic elements.

In the case of the present invention, the detectors and the layer arepreferably fixedly installed in a predetermined spatial arrangement in acommon housing. Since CCDs are insensitive to the energy spectrum of thephotons emitted by the radionuclides, no shielding is necessary and theCCDs can be completely integrated in the tomograph that is shieldedoverall.

Depending on the arrangement of the detectors, a highly reflective or adiffusely reflective layer is used in the case of the present invention.As a highly reflective layer, by way of example, aluminum isvapor-deposited onto a suitable base material having a low attenuationcoefficient. Such layers are available in the prior art. Diffuselyreflective thin layers are equally obtainable in the prior art, forexample in the form of micrograms of plastic applied to a suitable basematerial. In the case of the present invention, the layers are intendedto be as thin as possible in order to ensure a minimum attenuation andscattering of the radioisotopic photons, so that these effects arenegligible, in principle. If they are present at all, scattering andabsorption by the layer can be compensated for in an imagereconstruction following the detection. The minimum thickness isdetermined by the required static properties, for example the planarstiffness.

In a preferred embodiment of the present invention, the at least oneSPECT detector comprises an (e.g. planar) scintillation crystal arraywith a multiplicity of scintillation crystals and a spatially resolvingphotomultiplier array. The scintillation crystals are dense, transparentcrystalline materials (for example NaI(Tl)) that serve as converters forhigh-energy γ rays into visible light. The visible light is detected inthe form of electrical signals in spatially resolving fashion by thephotomultiplier array.

The present invention furthermore relates to an imaging method foralternately determining in vivo distributions of bioluminescent and/orfluorescent markers and in vivo distributions of radioactive markers bymeans of a common measurement setup at identical projection angles, thedistribution of the bioluminescent and/or fluorescent markers beingdetermined by separate detection of photons having a first averageenergy, which are emitted by the bioluminescent and/or fluorescentmarkers, by means of at least one first detector and, alternately withrespect thereto, the distribution of the radioactive markers beingdetermined by separate detection of photons having a second averageenergy, which are emitted by the radioactive markers, by means of atleast one second detector.

In order to carry out this method according to the invention, use ispreferably made of an apparatus in which the masks serving ascollimators for the SPECT detectors, during a measurement, can be movedout of the fields of view of the CCD cameras and can be moved into thefields of view again.

If the masks are in this case situated outside the fields of view of theCCD cameras, then the sensitivity of these optical imaging systems issignificantly increased. The detection of radioisotopic photons is notpossible, however, in this state (without collimation). Therefore, theSPECT detectors are preferably inactive in the case of masks removedfrom the beam path. If the masks are situated in the beam path, then theCCD cameras are preferably inactivated. Temporally alternatingintroduction of the masks into the two positions (within/outside thefield of view of the CCD cameras) results in a preferred applicationmode of the apparatus according to the invention.

The present invention is preferably used for in vivo studies of smallanimals (for example mice or rats), for in vivo observation of geneexpression and for breast, prostate, skin tumor and thyroid glandimaging.

The present invention is explained in greater detail below withreference to the drawing.

In the figures:

FIG. 1 shows a preferred embodiment of an apparatus according to theinvention for carrying out the imaging method according to theinvention,

FIG. 2 shows a schematic illustration of the spatial arrangement of thedetectors in a further possible embodiment of the apparatus according tothe invention,

FIG. 3 shows a further preferred embodiment of an apparatus according tothe invention with two CCD cameras and two SPECT detectors,

FIG. 4 shows a further embodiment of an apparatus according to theinvention with two CCD cameras and two SPECT detectors, and

FIG. 5 shows a modification of the embodiment of an apparatus accordingto the invention as shown in FIG. 3 with moveable masks, and

FIG. 6 shows a preferred modification of the embodiment of an apparatusaccording to the invention as shown in FIG. 1 with just one CCD camera.

FIG. 1 shows a preferred embodiment of an apparatus according to theinvention with two CCD cameras and one SPECT detector.

In the case of this preferred embodiment of the present invention, theapparatus according to the invention comprises two CCD cameras 1, 2facing one another, an SPECT detector 3 arranged perpendicular to theCCD cameras 1, 2, a shielding 4 arranged in front of the SPECT detector3 and bent at an angle of 90°, and a layer 5 fixed on the shielding 4and likewise bent at an angle of 90°, the bending edge 6 of said layerlying on the bending edge of the shielding 4, the layer 5 covering anaperture 7 in the shielding 4. The layer 5 reflects optical photons, forexample photons from bioluminescent and/or fluorescent markers, andtransmits γ photons, for example photons from radioactive markers. Theshielding 4 with the aperture 7 serves as a collimator for the SPECTdetector 3. The subject to be examined (in this case the mouse 8), whichis preferably situated in a thin-walled transparent tube 9 made ofPlexiglas, is arranged as near as possible in front of said collimator.If the mouse 8 contains radioactive and bioluminescent and/orfluorescent markers and if said markers emit photons in the direction ofthe shielding 4, then the photons are largely reflected at the layer 5or largely transmitted by the layer 5, depending on their energy. Threebeam courses 10, 11, 12 are depicted in FIG. 1 for low-energy, opticalradiation. The three beams 10, 11, 12 are reflected at an angle of 90°at the layer 5, with the result that they are steered directly to theCCD cameras 1, 2 and detected there. The medium- or high-energy γphotons can pass through the layer 5 without or with only slightinteractions. They are then either absorbed by the shielding (forexample made of lead or tungsten) or they pass through the aperture 7into the SPECT detector 3, where they are detected. The SPECT detectorcomprises a scintillation crystal array 13 and a photomultiplier array14, which transform the incident γ photons into optical photons andsubsequently into an electric current. The SPECT detector 3 with theshielding 4 and the layer 5 and the CCD cameras 1, 2 are arrangedfixedly in a housing 15, so that they maintain a specific spatialarrangement relative to one another. The housing 15 with the fixedlyarranged elements can be rotated about the tube 9 with the mouse 8,preferably through 360°, in order to obtain measurement data atdifferent angles. On the other hand, it is also conceivable to rotatethe tube 9 together with the mouse 8 in the fixedly arranged housing 15.

The projection solid angle in FIG. 1 is 0 degrees by definition. Theimaging planes of the detectors are parallel to one another (that is tosay have the same projection angle of 0 degrees), although the imagingplanes of the CCD cameras 1, 2 are rotated by respectively plus/minus 90degrees with respect to the imaging plane of the SPECT system. Identicalprojection angles are provided because the fields of view (photonprojection trajectories) of both CCDs 1, 2 experience a rotation through90 degrees (through the reflective layer 5).

FIG. 2 shows a schematic illustration of the spatial arrangement of thedetectors in a further possible embodiment of the present invention.

In this case, the design from FIG. 1 was adopted and also additionallyconstructed in mirrored fashion at an axial rotation axis runningparallel to the CCD camera longitudinal axis centrally through thesubject 16 that had been placed into a tube 9. Thus, photons emittedaccording to one direction can be detected by a first and a second CCDcamera 1, 2 and a first SPECT detector 20 and photons emitted in theopposite direction can be detected by a third and fourth CCD camera 17,18 and a second SPECT detector 19.

FIG. 3 shows a further preferred embodiment of an apparatus according tothe invention with two CCD cameras and two SPECT detectors.

In the case of this preferred embodiment of the present invention, theapparatus according to the invention comprises two CCD cameras 1, 2oriented in the same direction and spaced apart from one another; twoSPECT detectors 19, 20 arranged perpendicular to the CCD cameras 1, 2,two masks 23, 24 with at least two apertures 7 in each case and twolenses 25, 26 between the two SPECT detectors 19, 20. Furthermore, theapparatus contains two reflectors 27, 28 essentially comprising a layer5 in each case, which are oriented in such a way that they largelyreflect, in the direction of the CCD cameras, the photons that areemitted by the bioluminescent and/or fluorescent markers, aretransmitted through the apertures in the masks in the direction of theSPECT detectors and are focused by the lenses. The photons havingdifferent energy that are emitted by the bioluminescent and/orfluorescent and radioactive markers contained in the mouse 8 firstlyhave to pass through the masks 23, 24 with the apertures 7, said masksserving as SPECT collimators. Afterward, the optical photons are focusedby the lenses 25, 26 onto the layer 5 of the respective reflector 27,28, reflected at the highly reflective layer 5 in the direction of therespective CCD camera 1,2 and detected there. The beam course of twooptical beams 29, 30 is depicted in FIG. 3. The γ photons interactscarcely or not at all with the lenses 25, 26 and the reflectors 27, 28,so that they can propagate in the acceptance cone 31 of the apertures 7unimpeded in the direction of the scintillation crystal array 13 of therespective SPECT detector 19, 20 in which they are detected. Scatteringand absorption (if present) caused by the lenses 25, 26 and layers 5 canbe compensated for in a mathematical image reconstruction following theacquisition. The lenses 25, 26 preferably consist of materials having alow attenuation coefficient for the radioisotopic photons, for examplePlexiglas.

The entire rigid arrangement combined in the housing 15 (CCD cameras 1,2, reflectors 27, 28, lenses 25, 26, masks 23, 24 and SPECT detectors19, 20) can be rotated about the tube 9, preferably through 360°, inorder to be able to obtain a series of measurement data at identicalangular distances all around the subject to be examined.

In principle, only half of the apparatus illustrated in FIG. 3 alsosuffices for examining the mouse 8, that is to say a CCD camera 1, amask 23, an SPECT detector 19, a lens 25 and a reflector 27 with a layer5. The double arrangement shown in FIG. 3 has the advantage, however,that in the case of tomographic application of the design, thesensitivity of the camera system is twice as high for the sameacquisition.

The apparatus according to the invention as illustrated in FIG. 3optionally comprises a position sensor 35 for determining the currentposition of the mouse, which possibly moves in the tube 9 during ameasurement. An optical (standard) positioning system that continuouslyrecords the position of markers fitted externally to the mouse isinvolved in this case. This additional positioning system is necessaryonly when the mouse is permitted to move in the tube 9, since it is thennot possible to exactly derive (and compensate for) the movement of theanimal from the distributions acquired in vivo.

FIG. 4 shows a further embodiment of an apparatus according to theinvention.

In the case of this preferred embodiment of the present invention, theapparatus according to the invention comprises two CCD cameras 1, 2oriented parallel and oppositely to one another, between two SPECTdetectors 19, 20 facing one another and two masks 23, 24 with at leasttwo apertures 7 in each case, a respective layer 5 being situated infront of the SPECT detectors 19, 20, said layer largely reflecting thephotons emitted by the bioluminescent and/or fluorescent markers andlargely transmitting the photons emitted by the radioactive markers. Inthis case, the housing 15 is subdivided into two symmetricallyconstructed chambers 32, 33, in the center of which the subject (mouse8) to be examined is situated in a tube 9, the entire arrangementcombined in the housing 15 being mounted such that it can be rotated,preferably through 360°, about said subject. The masks 23, 24 aresituated on opposite sides of the mouse 8, a portion of the photonsemitted by the fluorescent, luminescent and radioactive markers passingthrough the apertures 7 of said masks into the acceptance cone 31 of theSPECT detectors. The photons of the radioactive markers pass largelywithout interaction through the layer 5 and are subsequently detected bythe SPECT detector 19, 20. The photons having lower energy from thefluorescent or bioluminescent markers are largely reflected at the layer5. The layer 5 preferably diffusely reflects the photons emitted by thebioluminescent and/or fluorescent markers, so that a portion of thereflected radiation is reflected in the direction of the CCD cameras 1,2 and detected there. The field of view 34 of the CCD cameras isillustrated in FIG. 4.

In principle, only half of the apparatus illustrated in FIG. 4 alsosuffices for examining the mouse 8, that is to say a CCD camera 1, amask 23 and an SPECT detector 20 with a diffusely reflective layer 5.The double arrangement shown in FIG. 4 has the advantage, however, of ahigher measurement signal overall since the photons emitted in bothdirections are detected and, consequently, a higher resolution of theimage is calculated therefrom.

FIG. 5 shows a modification of the embodiment of an apparatus accordingto the invention as shown in FIG. 3 with moveable masks.

In principle, the construction of this embodiment of an apparatusaccording to the invention corresponds to that shown in FIG. 3. Inaddition, in the case of this modification, the first and second masks23, 24 are fixed in such a way that, during the acquisition, they can belead out of the fields of view of the CCD cameras 1, 2 (from position Ato position B) and be put back into the initial position (A) again. Ifthe masks 23, 24 are situated outside the fields of view of the CCDcameras 1, 2 (position B), then the sensitivity of these optical imagingsystems is significantly increased. The detection of radioisotopicphotons is not possible, however, in this state (without collimation).Therefore, the SPECT detectors 19, 20 are inactive in the case of masks23, 24 that are removed from the beam path. Lenses are not required inthis embodiment of the present invention. The CCD cameras 1, 2 areprovided with an optical arrangement for focusing the beams emitted bythe bioluminescent and/or fluorescent markers. If the masks 23, 24 inthe embodiment of the apparatus according to the invention as shown inFIG. 5 are in position A, then the CCD cameras 1, 2 are preferablyinactivated. Temporally alternate introduction of the masks 23, 24 intothe positions A and B results in a further preferred application mode ofthe design from FIG. 3.

FIG. 6 reveals a preferred modification of the embodiment of anapparatus according to the invention as shown in FIG. 1 with just oneCCD camera.

The imaging principle as illustrated in FIG. 1 is adopted in the case ofthis preferred embodiment of the present invention. The construction andthe method of operation of the SPECT camera 3, including the shielding4, the aperture 7 and the arrangement and method of operation of thereflective layer 5, are identical to the apparatus described therein. Ina departure, however, in accordance with this embodiment variant, theseparate fields of view of the reflective layer 5 are defined by meansof an arrangement of mirrors 39, 40, 45 in such a way that they areimaged in a manner adjoining one another in the objective of the CCDcamera 38 and thus on the light-sensitive matrix. The mirrors 39 and 45are preferably configured in planar fashion, while the mirrors 40 may beformed such that they are curved concavely. The mirrors 45 aretransmissive on one side, so that laser beams can optionally beintroduced in order to stimulate NIRF markers.

This type of projection assembly can be chosen since the spatialresolution of the CCD camera 38 is at least one order higher than thephysically possible geometric resolution in the object, here the mouse8, which is limited on account of photon scattering processes in theobject 8. The advantage of this modification is an embodiment of thesubject matter of the present invention which is of spatially morecompact construction and less expensive.

In order to be able to optimally image animals of different sizes, inthe embodiment variant illustrated in FIG. 6, the SPECT camera 3including the shielding 4, the aperture 7 and the reflective surface 5and also mirrors 45 and laser beam coupling-in arrangements 43 ismounted on a platform 41 that can be displaced by means of a steppermotor 44. The field of view imaging of the CCD camera 38 is defined suchthat the average imaging lengths of the two half-fields from the object8 to the objective of the CCD camera 38 are of identical magnitude. Thiscondition is also ensured when the radial position of the displaceableplatform 41 with respect to the object 8 is changed. The platform 41including all of the partial apparatuses mounted thereon, such as, byway of example, the stepper motor 44 required for the drive, the mirrors39 and 40, the shielding 4 and also the CCD camera 38, are fixed on arotatable mounting support 42. The mounting support 42 can be rotatedthrough 360° by means of a stepper motor 46, so that theprojection-identical fields of view of both camera systems can bearranged at any desired common projection angle with respect to theobject 8 for the purpose of obtaining image data. While the mountingsupport 42 is accommodated in rotatable fashion on the housing 15, thestepper motor 46 is fixedly connected to the housing 15.

In all of the described embodiments of the present invention, theaperture or apertures is or are countersunk elongate opening(s). Thesecountersunk openings have the appearance e.g. of the detail enlargementA of FIG. 4. In this case, the apertures narrow from the outside 36 to aspecific diameter 37, which they then maintain as far as the center.This reduces the penetration of isotopic photons in the region ofconically tapering aperture edges.

LIST OF REFERENCE SYMBOLS

-   1 First CCD camera-   2 Second CCD camera-   3 SPECT detector-   4 Shielding-   5 Layer-   6 Bending edge of the layer-   7 Aperture-   8 Mouse-   9 Tube-   10 First beam course-   11 Second beam course-   12 Third beam course-   13 Scintillation crystal array-   14 Photomultiplier array-   15 Housing-   16 Subject-   17 Third CCD camera-   18 Fourth CCD camera-   19 Second SPECT detector-   20 First SPECT detector-   23 First mask-   24 Second mask-   25 First lens-   26 Second lens-   27 First reflector-   28 Second reflector-   29 First optical beam-   30 Second optical beam-   31 Acceptance cone of the SPECT detectors-   32 First chamber-   33 Second chamber-   34 Field of view of the CCD cameras-   35 Position sensor-   36 Narrowing of the aperture-   37 Constant diameter in the center of the opening-   38 Further CCD camera-   39 Mirror, planar-   40 Mirror, curved concavely-   41 Platform, radially displaceable-   42 Mounting support, rotatable-   43 Laser beam coupling-in arrangement-   44 1st stepper motor-   45 Mirror, transmissive on one side-   46 2nd stepper motor

1-21. (canceled)
 22. An imaging method for simultaneously determining invivo distributions of bioluminescent and/or fluorescent markers andradioactive markers at identical projection angles, the distribution ofthe bioluminescent and/or fluorescent markers being determined byseparate detection of photons having a first average energy, which areemitted by the bioluminescent and/or fluorescent markers, by means of atleast one first detector and the distribution of the radioactive markersbeing determined by simultaneous separate detection of photons having asecond average energy, which are emitted by the radioactive markers, bymeans of at least one second detector.
 23. The imaging method as claimedin claim 22, characterized in that the photons of the bioluminescentand/or fluorescent markers having the first average energy and thephotons of the radioactive markers having the second average energy areseparated for the separate detection with the aid of a layer, the layeressentially reflecting or transmitting the photons in a manner dependenton their energy.
 24. The imaging method as claimed in claim 23,characterized in that the layer serves for reflecting the photons of thebioluminescent and/or fluorescent markers in the direction of the atleast one first detector and for transmitting the photons of theradioactive markers in the direction of the at least one seconddetector.
 25. The imaging method as claimed in claim 22, characterizedin that the bioluminescent and/or fluorescent markers comprise at leastone marker from the group consisting of the markers of the luciferasereporters, the marker molecules having emission wavelengths in the nearinfrared range (NIRF molecules) and the molecules of the GFP (greenfluorescent protein).
 26. The imaging method as claimed in claim 22,characterized in that the radioactive markers comprise at least onemarker from the group As-72, Br-75, Co-55, Cu-61, Cu-67, Ga-67, Gd-153,1-123, I-125, I-131, In-111, Ru-97, Tl-201, Tc-99m and Xe-133.
 27. Theimaging method as claimed in claim 22, characterized in that thedetection of the photons having the first average energy is carried outby means of at least one CCD camera and the detection of the photonshaving the second average energy is carried out by means of at least onesingle photon emission computer tomography (SPECT) detector (3)comprising a collimator with at least one aperture.
 28. An imagingmethod for alternately determining in vivo distributions ofbioluminescent and/or fluorescent markers and in vivo distributions ofradioactive markers by means of a common measurement setup at identicalprojection angles, the distribution of the bioluminescent and/orfluorescent markers being determined by separate detection of photonshaving a first average energy, which are emitted by the bioluminescentand/or fluorescent markers, by means of at least one first detector and,alternately with respect thereto, the distribution of the radioactivemarkers being determined by separate detection of photons having asecond average energy, which are emitted by the radioactive markers, bymeans of at least one second detector.
 29. An apparatus for carrying outthe imaging method as claimed in claim 22, containing at least one CCDcamera at 1 st detector, at least one single photon emission computertomography (SPECT) detector as second detector and a layer, whichessentially reflects the photons of the bioluminescent and/orfluorescent markers and essentially transmits the photons of theradioactive markers.
 30. The apparatus as claimed in claim 29,characterized in that the at least one SPECT detector comprises ascintillation crystal array with a multiplicity of scintillationcrystals and a spatially resolving photomultiplier array.
 31. Theapparatus as claimed in claim 29, comprising two cooled CCD camerasfacing one another, a SPECT detector arranged perpendicular to the CCDcameras, a shielding arranged in front of the SPECT detector and bent atan angle of 90°, and a layer fixed on the shielding and likewise bent atan angle of 90°, the bending edge of said layer lying on the bendingedge of the shielding, the layer covering an aperture in the shieldingand largely reflecting the photons emitted by the bioluminescent and/orfluorescent markers and largely transmitting the photons emitted by theradioactive markers.
 32. The apparatus as claimed in claim 29,comprising two cooled CCD cameras oriented parallel and oppositely toone another between two SPECT detectors facing one another and two maskswith at least two apertures in each case, a respective layer beingsituated in front of the SPECT detectors, said layer largely reflectingthe photons emitted by the bioluminescent and/or fluorescent markers andlargely transmitting the photons emitted by the radioactive markers. 33.The apparatus as claimed in claim 32, characterized in that thediffusely reflects the photons emitted by the bioluminescent and/orfluorescent markers.
 34. The apparatus as claimed in claim 29,comprising two cooled CCD cameras oriented in the same direction andspaced apart from one another, two SPECT detectors arrangedperpendicular to the CCD cameras, two masks with at least two aperturesin each case and two lenses between the two SPECT detectors, tworeflectors essentially comprising a layer, which are oriented in such away that they largely reflect, in the direction of the CCD cameras, thephotons that are emitted by the bioluminescent and/or fluorescentmarkers, transmitted through the apertures in the masks in the directionof the SPECT detectors and focused by the lenses.
 35. The apparatus asclaimed in claim 34, comprising a position sensor for determining thecurrent position of a subject to be examined.
 36. The apparatus asclaimed in claim 34, characterized in that the masks, during ameasurement, can be moved out of the fields of view of the CCD cameras(position B) and can be moved into the fields of view (position A). 37.The apparatus as claimed in claim 31, characterized in that the apertureis a countersunk elongate opening.
 38. The apparatus as claimed in claim29, comprising a cooled CCD camera, a SPECT detector arrangedperpendicular to the CCD camera, a shielding arranged in front of theSPECT detector and bent away at an angle of 90°, and a reflective layerfixed on the shielding and likewise bent at an angle of 90°, the bendingedge of said layer lying on the bending edge of the shielding, thereflective layer covering an aperture in the shielding, the SPECTdetector, the shielding together with reflective layer and aperture andalso mirrors and laser coupling-in arrangements being accommodated on aplatform formed in displaceable fashion.
 39. The apparatus as claimed inclaim 38, characterized in that the displaceable platform is arranged ona mounting support formed in rotatable fashion.
 40. The apparatus asclaimed in claim 38, characterized in that separate fields of view ofthe reflective layer are imaged in a manner adjoining one another bymeans of an arrangement of mirrors in the objective of the CCD camera.41. The apparatus as claimed in claim 38, characterized in that laserbeam coupling-in arrangements and also mirrors that are transmissive onone side are accommodated opposite one another on the displaceableplatform in order to excite NIRF markers in the object by means of laserbeams.